The invention relates to a specific method for the assay of heparin.
Heparin is an inhibitor of the thrombin-fibrinogen blood clotting system and is used in patients undergoing procedures in which the prevention of clot formation is important, for example, patients undergoing extracorporeal blood circulation and/or treatment.
The level of heparin in whole blood can be measured in a number of ways, the simplest being a measurement of the activated clotting time (ACT), (Hattersley, 1966 J.A.M.A. 196:436). This method measures the time that it takes for blood, treated with diatomaceous-earth, to clot. There is a straight line relationship between the amount of physiologically active heparin in the blood and the delay in the time taken for a clot to form (Bull et al. 1975, J. Thorac. Cardiovasc. Surg. 69:685). Most types of cardiac surgery currently performed, including about 400,000 coronary bypasses per year in North America, require high levels of heparinization of the patient and an extracorporeal oxygenation circuit during cardiopulmonary bypass. Heparin reversal is routinely carried out with intravenous protamine sulfate at the end of the procedure, and the patient's coagulation status is followed by monitoring the activated clotting time. A number of complications with the use of protamine have been described, including fatal reactions. Heparin reversal is not always complete by this method, and may require additional protamine in the intensive care unit; the phenomenon of "heparin-rebound" has also been described where heparin activity is mobilized from fat stores during rewarming of the patient in the hours following surgery, and may contribute to excess bleeding during this period. A second method entails the chemical measurement of heparin. this method makes use of the chromophore, azure A (Klein et al., 11982, Anal. Bioc. 124:59). Unlike physiological assays this chemical assay is not affected by the presence of other anticoagulants, but detects both physiologically and non-physiologically active fragments of heparin and so is not adequate for managing heparin and protamine therapy during or after extracorporeal circulation or treatment of blood.
The analysis of certain other factors affecting blood clotting has been described by Hutt et al. 1972, J. Lab. Clin. Med. 79:1027, where partially purified heparinase was added to plasma samples prior to performance of the prothrombin and partial thromboplastin times. Elimination of the heparin effect was demonstrated for these coagulation tests, but the use of the enzyme was not extended to any other measures of coagulation status. The authors conclude their paper with the statement:
"The availability of this reagent will make possible the detailed investigation of coagulation mechanisms in severely ill, hospitalized patients receiving heparin therapy. Specific clinical situations in which the use of heparinase would be useful are: (1) the patient with disseminated intravascular coagulation already being treated with heparin; and (2) the patient with myocardial infarction or venous thromboembolic disease being changed from heparin to coumadin therapy. It is entirely possible that a standardized amount of heparinase could be added to commercially available coagulation assay reagents on a routine basis, thus avoiding inaccuracies resulting from unsuspected plasma contamination with heparin."
The coagulation tests and the methods described in this report are not applicable to the needs of cardiovascular surgery. Heparinase has also been used for the deheparinization of blood before it is returned to a patient (Langer et al., 1982, Science 217:261).
Although the heparin-ACT does-response method has markedly improved heparin and protamine therapy in cardiovascular surgery, certain problems remain. Despite complete heparin reversal at the end of the surgical procedure, some patients develop heparin-related coagulation defect in the early (up to 8 hours) postoperative period, referred to as "heparin rebound" (Ellison et al. 1974 J. Thorac. Cardiovasc. Surg. 67 723-729.). The ACT will be prolonged, and specific therapy requires more protamine. On the other hand, cardiopulmonary bypass and extracorporeal circulation may induce a number of secondary coagulation defects that are not heparin-related. Such defects may be caused by hemodilution, hypothermia, platelet disorders, disseminated coagulation, or fibrinolysis, and they may appear as a bleeding tendency in the operating room or in the postoperative period. Because the ACT is prolonged by these disorders, the bleeding tendency may be attributed to heparin; however, these secondary coagulation defects are not protamine-reversible. Specific and rapid determination of heparin activity is not available, and most patients are treated in a "shotgun" manner with protamine and blood products in this situation. The well known risks of protamine treatment make specific identification of secondary coagulation defects a highly desirable aim.